Measurement of chlorophyllase activity in olive fruit (Olea europaea).

Chlorophyllase activity in olive fruit (Olea europaea) was measured using the enzyme solubilized from the protein precipitate. The reaction was stopped by freezing the mixture at -20 degrees C, to avoid dilution of the sample and consequent reduction of the substrate levels to below the detection limits of the analytical system. Separation of the substrates and products of the enzymatic reaction was performed by reverse-phase HPLC using a gradient solvent system of water and ion suppressor/methanol/acetone. These conditions allowed direct resolution of the reaction mixture prior to centrifugation, without the need for the transfer of any of the components to other solvents. Olive chlorophyllase in the crude enzymatic extract showed maximum activity at 50 degrees C and the optimum pH was 8.5 in acetate-phosphate-borate buffer for all substrates used, chlorophylls (a and b) and pheophytins (a and b). The Km and Vmax values obtained for hydrolysis of these substrates showed that chlorophyllase had a greater affinity for chlorophyll b, while the highest maximum rate of reaction occurred with pheophytin a. Substrate inhibition was observed with pheophytin b.

Chlorophyll and carotenoid patterns in olive fruits, Olea europaea Cv. arbequina.

In olive fruits of the cultivar Arbequina, the chlorophyll pigments decrease significantly throughout ripening, while the carotenoids decrease more gradually and discontinuously. There is no degradation of the carotenoid fraction in stages before complete ripeness. The presence of esterified xanthophylls exclusively in this variety suggests that the chloroplast pigment metabolism is different from that in other olive varieties studied previously. There are increases of specific carotenoids, violaxanthin, neoxanthin, antheraxanthin, lutein epoxide, and esterified xanthophylls between the light green and yellowish green ripening stages. Such increases are related to the detection of precursor carotenoids (phytofluene and xi-carotene) in the yellowish green stage. Chlorophyllides (a and b) and alpha-carotene have also been detected exclusively in this variety. Quantitatively, the drastic change in color between light green and yellowish green ripening stages characteristic of this variety can be explained by the considerable reduction found in the chlorophylls/carotenoids ratio. The study of the pigments present in skin and pulp has shown that the pattern of carotenoid distribution differs depending on the fruit part concerned.

Chlorophyll pigment composition in table olives (cv. Gordal) with green staining alteration.

Metallochlorophyllic complexes of copper are present in green table olives, showing the alteration known as green staining. They were formed stepwise in such a way that new metallochlorophyll derivatives were detected as the fruits became more altered. Cu-15-glyoxylic acid pheophytin a was the first compound formed, followed in order by Cu-pheophytin a, Cu-15-glyoxylic acid pheophytin b, Cu-15-formyl-pheophytin a, and Cu-pyropheophytin a. Pigment analysis in fruits classified according to the surface area affected by green staining showed a progressive increase in the concentration of all copper complexes with the course of the alteration. The metallochlorophyll derivatives of copper were present both in the part of the fruit affected by the alteration and in the remainder part, although the amount was significantly greater in the former. On the other hand, not all the copper present in the fruits was accessible for the formation of such compounds, since the concentration of copper complexes found in olives with the maximum degree of alteration observed did not exceed 20% of the total copper of the fruit.

Identification of oxidized chlorophylls and metallochlorophyllic complexes of copper in table olives (cv. Gordal) with green staining alteration.

In Spanish green table olives showing the alteration known as green staining, new chlorophyll derivatives have been identified: the copper complexes of 3(1),3(2)-didehydrorhodochlorin-15-glyoxylic acid-17(3)-phytyl ester a and b (Cu-15-glyoxylic acid pheophytins a and b), and 3(1),3(2)-didehydro-15(1)-hydroxy-15(1)-hydroxyrhodochlorin- 15-acetic acid delta-lactone-15(2)-methyl-17(3)-phytyl ester a and b (Cu-15(1)-OH-lactone-pheophytins a and b). These compounds were isolated by normal phase thin-layer chromatography and identified from their UV-visible and mass spectra and by co-chromatography with authentic standards. The chromatographic and spectroscopic characteristics and the molecular mass for a new allomerized chlorophyll derivative and its copper complex are reported. The corresponding molecular structure according to the molecular mass has been proposed. The characterization of the latter compounds enables their possible detection in processed fruits and vegetables. The present work gathers new advances in the study of the color alteration in table olives and this is the first time that copper complexes of oxidized chlorophylls are detected in foodstuffs.